Polymerase chain reaction (PCR) is not restricted to DNA templates. RNA may also be amplified by PCR, because Taq polymerase possesses weak reverse transcriptase activity [1]. Since nonspecific PCR has been successfully exploited as a fingerprinting method and as a tool for polymorphism detection in genomic DNA [2,3], we have made use of nonspecific PCR to investigate alterations in gene expression in congenital hemolytic anemia.
The cellular integrity of the red blood cells depends on proteins that belong to four distinct functional systems: responsible for the repair of oxidative damage (pentose phosphate shunt-glutathione pathway), the system responsible for the transport of oxygen (hemoglobins), the system responsible for the cellular exchanges (membrane and cytoskeleton), and energy generation (glycolytic pathway). In each of these systems, mutations have been found that are responsible for hereditary hemolytic disorders. Among others, hereditary non-spherocytic anemia may be caused by deficiency of pyruvate kinase in the glycolytic pathway [4]. Hemolytic anemia occurs whenever the steady-state normal number of red cells is diminished, due to decreased stability. Since the bone marrow has the capability to increase its production of erythrocytes [5], hemolytic anemia is characterized by both increased hemolysis and increased erythropoiesis. Hence, the higher turnover rate of red blood cells is indicated by reticulocytosis and elevated bilirubinemia.
Using nonspecific PCR, we have obtained evidence that the expression of several genes is activated in blood cells four from patients with hereditary hemolytic anemia. Three are diagnosed with pyruvate kinase deficiency and one is of unknown cause. Two markers have been already identified and have been proposed to be human markers associated with hereditary hemolytic anemia [18]. We present here the genetic expression studies of marker B. This marker shows homology with bovine interferon .alpha.-A gene. Since interferon has a role as a differentiation factor in the erythroid cell lineage and is used as a therapeutic agent to treat hairy cell leukemia, marker B may be another related interleukin which may be used to develop pharmaceutical agents to be advantageously used in the treatment of hemolytic anemia caused inter alia by pyruvate kinase deficiency. This disease does not respond well to the well-known hematopoiesis growth factor, erythropoietin [21]. Marker A is, on the other hand, homologous to platelet-derived growth factor receptor (PDGFr). We still do not know if marker A is part of a DNA sequence encoding a receptor which responds to marker B or if marker A responds to another growth factor, like PDGF.
Even though the above two genes were identified as markers of hemolytic anemia, no specific role has been assigned thereto.